1. Field of the Invention
The present invention relates to Coryneform bacteria containing plasmids made by recombinant DNA techniques carrying genetic information useful for the fermentative production of threonine and isoleucine.
2. Description of the Prior Art
It has been known in the prior art that in order to render a wild strain capable of producing L-threonine from carbohydrates it has been necessary to induce artificial mutants from the wild strain. There are many known L-threonine producing artificial mutants, most of which are resistant to .alpha.-amino-.beta.-hydroxyvaleric acid (hereinafter referred to as AHV), and belonging to the genus Brevibacterium or Corynebacterium. These microorganisms produce L-threonine in a yield of from 10 to 20%. For example U.S. Pat. Nos. 3,582,471, 3,580,810 and Japanese Publication No. 47-34956 describe threonine producing mutants resistant to AHV and belonging to the genera Brevibacterium, Escherichia and Corynebacterium. Threonine production by mutants of the genera Brevibacterium and Corynebacterium is also described in Japanese Published Unexamined Patent Applications Nos. 51-54984, 53-101591, 54-32693, 54-35285, 54-35286, 54-35288, 54-37886 and 54-92692.
U.S. Pat. No. 4,278,765 and Japanese Published Unexamined Patent Applications Nos. 55-131397 and 56-15696 describe and discuss threonine producing Escherichia coli strains transformed with a recombinant plasmid DNA. Commonly assigned copending U.S. patent application Ser. No. 376,396 filed May 10, 1982 at the U.S. Patent and Trademark Office describes the production of L-threonine with Coryneform bacteria harboring a plasmid having inserted therein a chromosomal DNA fragment controlling resistance to AHV.
The situation with L-isoleucine is very similar to that of threonine. Examples of known L-isoleucine producing microorganisms include mutants of Serratia resistant to isoleucine hydroxamate (Japanese Published Examined Patent Application No. 30593/1977), mutants of Corynebacterium glutamicum requiring L-leucine for growth (Japanese Published Examined Patent Application No. 38995/1972), mutants of Brevibacterium and Corynebacterium resistant to AHV (Japanese Published Examined Patent Application No. 2880/1965), mutants of Brevibacterium resistant to AHV and requiring lysine for growth (Japanese Published Examined Patent Application No. 6237/1976), mutants of Brevibacterium resistant to AHV and O-methylthreonine (Japanese Published Examined Patent Application No. 21077/1976), mutants of Corynebacterium resistant to S-(2-aminoethyl)-cysteine (Japanese Published Examined Patent Application No. 4629/1977), mutants of Escherichia resistant to 2-amino-3-methylthiobutyric acid (Japanese Published Unexamined Patent Application No. 69881/1978), and mutants of Brevibacterium resistant to AHV and trichloroalanine (Japanese Published Unexamined Patent Application No. 35287/1979).
The prior art has also described Escherichia coli strains transformed with a recombinant plasmid DNA, which strains have increased productivity of amino acids (See, for example, U.S. Pat. No. 4,278,765). It has generally been difficult, however, to construct commercially useful threonine (Thr) or isoleucine (Ile) producers of Escherichia coli by gene splicing techniques, because the original Escherichia strains do not express high productivity for threonine and isoleucine, and recombinant strains derived from such Escherichia strains do not produce high amounts of either amino acid.
On the other hand, there are many strains in the genera of Brevibacterium and Corynebacterium which produce high amounts of L-threonine and L-isoleucine. Strains of Corynebacterium and Brevibacterium may thus be suitable as original strains for construction of L-threonine and L-isoleucine producers by gene recombination techniques.
Although the presence of plasmids in strains of Brevibacterium and Corynebacterium having detectable phenotypic markers has not been known for a long time (but see, e.g., Published European Patent Application No. 003391), recent work has demonstrated the feasibility of preparing Coryneform bacteria harboring plasmids controlling the production of threonine or isoleucine (See the aforementioned commonly assigned, Ser. No. 376,396, copending at the U.S. Patent and Trademark Office filed May 10, 1982 (Thr), and commonly assigned copending Ser. No. 392,145, filed June 25, 1982 at the U.S. Patent and Trademark Office (Ile)). In addition, commonly assigned copending U.S. patent application Ser. No. 386,980 filed on June 10, 1982 at the U.S. Patent and Trademark Office, describes composite plasmids capable of propagating in Coryneform glutamic acid producing bacteria. (All of these patent applications are herein fully incorporated by reference).
A brief schematic representation of the isoleucine and threonine biosynthetic pathway is as follows: ##STR1##
In Scheme I is also shown part of the lysine biosynthetic pathway. The first branch of the pathway leading to threonine and isoleucine can be found at the junction of aspartate semialdehyde. The enzyme leading to these two amino acids is homoserine dehydrogenase (hereinafter "HDase"), while the enzyme leading to lysine is dihydrodipicolinate synthetase, ("DHDP synthetase").
The relationship between homoserine dehydrogenase activity and resistance to AHV in mutants of Corynebacteria is disclosed in Shiio et al Journal of Biochemistry 68: 859 (1970), Nakamori et al Agric. Biol. Chem. 37: 653 (1973), and Tosaka et al ibid 43: 265 (1979).
It should also be noted here that, recently, Escherichia coli strains were described which carry hybrid plasmids containing several genes of the lysine biosynthetic pathway. An overproducer of lysine (TOC R 21) was transformed, and the synthesis of lysine was studied in different strains (See, for example, LeReverend et al, European Journal of Applied Microbiology and Biotechnology, 15: 227-231 (1982), as well as published French Patent Application No. 2511032 (Application No. 81/15385) published Feb. 11, 1983). It appears from these publications that only plasmids containing the dapA gene (encoding DHDP synthetase) lead to an increase in lysine production; this reaction is the limiting biosynthetic step in lysine overproducers (having mutations altering the aspartokinase reaction). The authors suggest that such a method of gene amplification could be used to improve strains which overproduce metabolites. There is no suggestion in these publications, however, to expand this work to Coryneform bacteria or to any other products than lysine.
A need therefore still exist for improved and efficient methods for the fermentative production of L-threonine and L-isoleucine in Coryneform bacteria.